Infection of bats with Histoplasma species

Abstract Histoplasma species infect humans and animals, notably bats. Histoplasma species are thermally dimorphic fungi existing in mycelial form in the natural environment and in yeast form in infected tissues. In this narrative literature review, we summarize the occurrence of Histoplasma spp. in different species of bat tissues (n = 49) and in soil admixed with bat guano where the species of bat dwelling nearby has been identified (an additional 18 species likely infected) to provide an up-to-date summary of data. Most positive isolations are from the Americas and Caribbean, with some studies from Thailand, Malaysia, Nigeria, Slovenia, France, and Australia. We also summarize some of the early experimental work to elucidate pathogenicity, latency, immune response, and faecal excretion in bats. Given the recent recognition of the global extent of histoplasmosis, thermal dimorphism in Histoplasma spp., and global heating, additional work on understanding the complex relationship between Histoplasma and bats is desirable.


Introduction
A genus of pathogenic fungi shared between humans and bats is Histoplasma spp.Initial work on the natural habitat of Histoplasma spp.5][6] The first isolation from a bat ( Chilonycterus rubiginosa fusca ) came from Panama in 1962. 7Histoplasma duboisii has only been isolated once from bats in Nigeria. 8istoplasma capsulatum is a thermally dimorphic saprobic fungus that produces microconidia and macroconidia borne on mycelium. 9 , 10When suitable microclimatic conditions of temperature (22 • C-29 • C) and annual rainfall of 35-50 inches are present, and soil is enriched with avian excreta or bat guano, the fungus proliferates abundantly.Birds don't get infected with H . capsulatum , whereas bats can be infected and can disperse the fungus creating new foci of infection. 11Infection is acquired by inhalation of microconidia or hyphal fragments of H . capsulatum from environmental sources such as soil or dried avian or bat excreta.The rapidly changing global environment will inevitably alter some of these interactions.
Following inhalation by a human, the infectious propagules of H . capsulatum are engulfed by pulmonary macrophages, wherein they convert to the yeast form of the fungus and multiply and traverse to hilar and mediastinal lymph nodes. 9 , 10he yeast form of the fungus then gets access to the blood circulation system and disseminates swiftly across various organs of the body.After approximately two weeks of exposure, the macrophages become fully fungicidal, and cellular immunity increases, with defense against the fungal particles. 10This process can lead to necrosis at the sites of infection in the lungs, liver, spleen, lymph nodes, bone marrow, adrenal glands, and mucocutaneous membranes, and acute, sub-acute, or chronic disseminated histoplasmosis, acute pulmonary histoplasmosis, or latency.The human pathogenic life cycle of H . duboisii is less well understood. 12 , 13][16][17] Investigations by several workers have shown that bat guano and bird droppings are the most common sources of Histoplasma capsulatum , the etiological agent of histoplasmosis, which has a worldwide distribution. 11 , 18-20The fungus is found either in spaces where bat guano is abundant or in open spaces such as public parks and home yards, where avian droppings are frequently found.Bat and avian excreta provide nutrients necessary for fungal growth and with soil and environmental conditions, humidity, and temperature, constitute the ecologic niche of this microorganism. 1 , 19 , 21 Bats are among the few infected mammals that contribute to the maintenance of this fungus in natural foci.Our aim was to review the species of bats that have been reported to be infected by Histoplasma spp.and to link those reports with the bats' locations.The last time this topic was reviewed was in 1981 by Hoff and Bigler. 18

Materials and Methods
An exhaustive search of the literature, using PubMed, MED-LINE, Med Facts, and LILACS (a database Latin American and Caribbean Health Sciences Literature) was made for all years to September 10 th 2022 using different sets of keywords, viz.histoplasmosis, bats, caves, Histoplasma capsulatum var, capsulatum , Histoplasma capsulatum var.duboisii .The Boolean operator 'AND' was used for combining and narrowing the searches.Cross-references were also accessed to extract information relevant for incorporation into the review.Review of papers that were identified in searches also identified references that were not listed online and these were retrieved.No language restriction was applied.

Results
We identified 45 articles describing the isolations of Histoplasma spp.from bats, or detection using histopathology, polymerase chain reaction (PCR) or antigen.A smaller number or articles linked specific bat species to outbreaks in humans, providing inferential evidence of individual bat species infection.The data collected and the important findings found in the survey of literature are described below under different subheadings.

Occurrence of Histoplasma duboisii in bats and environmental sources
The only bat isolation of Histoplasma duboisii is from Nigeria in Nycteris hispida (hairy slit faced bat with long ears) and Tadarida pumila (little free-tailed bat) (Table 1 ).This was from intestinal contents only of a single bat, but it is not clear which species.Histological examination of the tissue of liver, spleen, and lung from this particular bat and 8 additional bats of Nycteris hispida and Tadarida pumila did not reveal the presence of Histoplasma nor were the cultures from the internal organs of any of the bats examined positive for Histo-plasma yeast forms.It is therefore unclear whether this fungus can infect bats.

Isolation of Histoplasma capsulatum directly from bats
We have tabulated the species of bats from which H. capsulatum has been directly isolated (Table 2 ; Supplementary Table 1).We found direct evidence of isolation from bat tissues in 48 species, all but one from the Americas and Caribbean, in studies from 1962 to 2021.Some bat species were sampled and cultures were negative (Supplementary Table 1).Attempts to isolate H. capsulatum from bats in Iran 44 and Zaire (now Democratic Republic of Congo) 45 were negative.In some studies, the bat species was not determined.These mostly culture-based studies have almost always included analysis of liver and spleen, usually lungs and sometimes intestines and/or intestinal contents.One study of 122 bats randomly captured in Argentina, French Guyana, Mexico found 77% positive by PCR of the lungs only, 46 and a single Nyctalus noctule bat in France was also positive by PCR from lungs. 38The assumption is that if bat tissues are positive, then the bat is infected.Given the pathology of the intestine showing clumps of yeast and concurrent positive cultures from the intestine demonstrated in Pteronotus ( Chilonycteris) rubiginosa , 28 , 47 this seems a reasonable assumption given the diets of bats.Some bats have histological evidence of infection in various tissues, with negative cultures, others positive cultures, without evidence of fungi histologically. 44Such studies have only been done in a limited number of bat species.The relative proportion of organs that were culture positive in several species of bats captured in SE USA (primarily Myotis austroriparius and Myotis grisescens ) were lungs (88%), spleen (73%), liver (72%), and faeces (36%). 38Recent studies from Brazil have limited sampling to the lungs and only used PCR for detection. 24 , 26 , 32xperimental infections using intraperitoneal inoculation demonstrated mice to be infectable with very few or a single spore 14 , 48 and bats were experimentally infected via both intranasal and intraperitoneal routes with inocula as low as 10 infective units intraperitoneally and 100 intranasally. 15-17 , 46xperimental infection found differences between species.So Tadarida brasiliensis had an 80% mortality whereas Artibeus lituratus and Pteronotus suapurensis were infectable, but without any mortality. 18Experimental intranasal infection of the lungs of A. lituratus was possible with fewer than Table 2. Bat species and country from which Histoplasma capsulatum has been isolated.T his e x cludes soil and guano samples, e v en if the roosting bat species was identified.

Artibeus hirsutus
Hairy fruit-eating bat only known in Ecuador and Peru Mexico T aylor, 1999 22 ; T aylor,

Leptonycteris nivalis
Greater long-nosed bat living in desert scrub areas Mexico T aylor, 1999 22

Mollosus rufus ( Molussus afer )
Black mastiff bat found throughout Central and South America.Can be found in dark places like rock fissures, in caves, roofs and cracks of buildings Mexico, Brazil Taylor, 2012 23 Veloso,

Mormoops megalophylla
Ghost-faced bat typically found near desert shrubs in Central and South America Mexico Taylor, 1999 22 Myotis austroriparius Southeastern myotis feeds on a variety of insects, living close to water sources Florida, USA Tesh, 1967 38 ; Hoff, 1981 18 Myotis californicus Californian myotis found in caves, mines, rocky hill sides, under tree bark, on the ground, and in buildings

Mexico
Taylor, 1999 22 Myotis grisescens Gray bat that occurs in a limited range in limestone areas of Southeastern USA USA Tesh, 1967 38

Vampyriscus bidens
Bidentate yellow-eared bat Brazil Da Silva, 2020 32 * presumptive identification 100 viable spores, 49 and led to disseminated infection, antibody production, and temporary cutaneous delayed hypersensitivity.Subsequently yeasts were shed via the intestinal villi into the faeces.Naturally infected bats of the species have 10-2000 viable Histoplasma cells in their faeces, when the total faecal contents were sampled. 27urther supportive evidence of past infection using Histoplasma antibody detection in bats was sought in several studies and negative in all samples. 25 , 27 , 50The immune response to natural infection by Histoplasma spp. in bats seems muted. 44

Isolation of Histoplasma capsulatum from soil and guano linked to bats
Indirect evidence of bat infection by Histoplasma spp. was sought by sampling and culturing guano from bat caves and roosts, and soil from these locations.In 10 studies, guano from the same bat species were found to be infected as from the bats themselves (Table 3 ).An additional 18 species of bats were presumptively infected, including four species of Hipposideros , based on guano alone (Table 3 ).Isolations of H. capsulatum were reported from multiple countries, including Puerto Rica, Thailand, Trinidad, Panama, Malaysia, Australia, Slovenia and the USA.

Outbreaks of histoplasmosis associated with bat guano, implicating species of bats
An outbreak of histoplasmosis is defined as the occurrence of at least two cases of the disease.Numerous reports of epidemics or outbreaks of histoplasmosis in the Americas have been described over the years, dating back to the late 1930s. 19 , 60 , 61To our knowledge, only one of these reports identified the species of bats linked to exposure-in Costa Rica.In this report, 15 students entered a cave in Santa Rosa National Park, Guanacaste Province inhabited by about 500 bats. 62Twelve of the students subsequently developed persistent illness, with abnormal chest radiographs and serologic evidence of acute pulmonary histoplasmosis.The cave housed three species of fruit bats ( Glossophaga soricina, Carollia perspicillata , and Carollia subrufra ) and one species of vampire bats ( Desmodus rotundus ).Staffolani et al. (2018) in an exhaustive review of literature identified 814 persons who acquired acute or disseminated histoplasmosis amongst the travellers to bat caves in different countries in the Caribbean, South America, some parts of North America, Europe, and Africa. 63These authors also mentioned details of exposure to bats, cases occurring in clusters, and types of antifungal therapy if given.They also provided a timeline distribution of the studies reporting cases of acute histoplasmosis in immunocompetent travellers.

Discussion
In 1981, Hoff and Bigler counted 24 species of bats with documented Histoplasma infection recorded (two not confirmed). 18We identified an additional 25 bat species infected, with indirect evidence of infection of another 18 species (total of 67).There are over 1400 species of bats present worldwide.The vast majority of studies of Histoplasma infection Table 3. Histoplasma -positive guano and soil from bat roosts implicating a particular species of bat as infected.

Species Common name and additional data Country References
Artibeus jamaicensis parvipes Jamaican fruit-bat prefers humid and tropical habitats but is also adapted to cloud forests and drier tropical areas Puerto Rica Torres-Blasini, 1966 51 Aselliscus stoliczkanus Stoliczka's trident bat found across SE Asia Changmai, Thailand Norkaew,

Carollia perspicillata
Seba's short-tailed bat found in moist evergreen and dry forests usually below 1 000 m elevation  64 It is highly likely that these and other areas have resident and migratory bat populations with histoplasmosis.Not only are bat and environmental data on Histoplasma missing from these and other areas, but reports of outbreaks of acute pulmonary histoplasmosis from most of these areas are also absent, the exceptions being New Caledonia, Indonesia, and South Africa. 63axonomy of the Histoplasma genus has recently undergone a revision with H. capsulatum var capsulatum being split into four species: H. capsulatum sensu stricto (also known as the Panama or H81 lineage), Histoplasma ohiense , H. missis-sippiense , and H. suramericanum . 65This taxonomic revision has yet to be fully accepted.It will take some time to ascertain if certain bat species are more or less likely to harbour any of these species/varieties/taxonomic groups preferentially.One bat from Sao Paolo state in Brazil was infected with H. suramericanum , assuming this is a valid species name. 66Recently sequence data from a small number of H. capsulatum isolates from bats in Mexico and migratory bats has started to unravel these relationships. 36hile bats may have Histoplasma spp.isolated from deep tissues, notably lungs, liver and spleen, and may be experimentally infected with low inocula, it is not clear to what extent they suffer from disease, or whether they have latent infection, with gastrointestinal colonization or excretion.Pathologic lesions were seen a few examined specimens of Pteronotus ( Chilonycteris) rubiginosa , but not many.It is probably unwise to generalize from one species to all others, especially where attempts to isolate Histoplasma spp.from multiple other bats was unsuccessful (Supplementary Table 1).Likewise given the variable manifestations and yield from these prior experiments (which often used mouse inoculation to improve culture sensitivity), it would be unwise to assume that some species of bat were intrinsically immune to infection with Histoplasma spp.A complicating factor in understanding Histoplasma infection in bats is their variable temperature over time.There is probably some variation in temperature in different species, but body temperature during flight is typically 40 • C, and it falls to lower than 10 • C when in torpor. 67These temperatures bracket the dimorphism temperature sensitivity of Histoplasma spp, typically around 32 • C, when filamentous forms at the lower temperature shift to a yeast form.To add further complexity, some strains of H. capsulatum grow more slowly at higher temperature, notably above 39 • C. 68 To our knowledge the biological implications of the temperature dynamics of bats on the physiology of Histoplasma spp .have yet to be fully addressed.Changing weather patterns, extremes of temperature and possibly altered migratory patterns of bats in response to these climatic pressures, could alter the 'status quo' between bats and Histoplasma spp..The recent outbreak of the lethal fungal infection of bats in N. America of White Nose Syndrome, [69][70][71] has led to a resurgence of interest in bat disease.Further attempts are underway to immunize bats to prevent mass die-offs and potentially species extinctions. 72It is conceivable that Histoplasma infection of bats could evolve and become problematic for bats, humans and the other mammals that can be infected.Infection due to H . capsulatum in cats and dogs is reported to be frequent in Midwest and southern United States. 73A recent survey of Histoplasma in mammals in Europe indicates a much wider endemic area than previously appreciated. 74

Conclusion
This study provides an update on the occurrence of Histoplasma spp. in bats and soil mixed with bat guano.Fifty four of the approximately 1400 species of bat have been documented to be infected by Histoplasma spp.There has been slow progress in this area in recent decades.There is a need to explore the occurrence of histoplasmosis in human populations around caves that harbour bats in areas of endemicity of histoplasmosis.Only one bat species has been reported to be infected with H. duboisii (in Nigeria), yet this fungus is present throughout the African continent.A better understanding of the pathogenicity , latency , and intestinal excretion of Histoplasma in bats is called for.

Table 1 .
Association of H. duboisii with bats.